Control of aggregation in protein refolding: The temperature‐leap tactic

Open Access
Publisher Website
Google Scholar
Abstract
The kinetics of renaturation of bovine carbonic anhydrase II (CAII) were studied from 4° to 36°, at the relatively high [CAII] of 4 mg/mL. Following dilution to 1 M guanidinium chloride, aggregate formation is very rapid and reduces the formation of active enzyme. The CAII activity yield at 150 min, 20° (∼60%), is greater than that at either 4° or 36°. However, if refolding is conducted at 4°, aggregation is reduced dramatically and 37% yield is obtained at 120 min. If the solution is then rapidly warmed to 36°, the yield rises rapidly to 95% at 150 min. This is an example of the “temperature leap” tactic. These results can be understood on the basis of two slow-folding intermediates whose kinetics have been studied. Only the first of these forms aggregates. Kinetic simulations show that, at 4°, the first intermediate is depleted after 120 min, and the second intermediate rapidly isomerizes to active enzyme on warming. A series of experiments was conducted where the initial (120 min) folding temperature was systematically varied, followed by a “leap” to 36° for 30 additional minutes. With initial incubations from 4° to 12°, the final yield is >90%, drops rapidly from 12° to 20°, and decreases more gradually to ∼45% at 36°. The overall results qualitatively fit the simple idea of ordinary temperature-accelerated reactions in competition with hydrophobic aggregation, which is strongly suppressed in the cold. Qualifications are discussed for the temperature-leap approach to find application in refolding other proteins.
Funding Information
  • College of Arts and Science of the University of Delaware